Anti‐tumoural activity of the G‐quadruplex ligand pyridostatin against BRCA1/2‐deficient tumours

Abstract The cells with compromised BRCA1 or BRCA2 (BRCA1/2) function accumulate stalled replication forks, which leads to replication‐associated DNA damage and genomic instability, a signature of BRCA1/2‐mutated tumours. Targeted therapies against BRCA1/2‐mutated tumours exploit this vulnerability by introducing additional DNA lesions. Because homologous recombination (HR) repair is abrogated in the absence of BRCA1 or BRCA2, these lesions are specifically lethal to tumour cells, but not to the healthy tissue. Ligands that bind and stabilise G‐quadruplexes (G4s) have recently emerged as a class of compounds that selectively eliminate the cells and tumours lacking BRCA1 or BRCA2. Pyridostatin is a small molecule that binds G4s and is specifically toxic to BRCA1/2‐deficient cells in vitro. However, its in vivo potential has not yet been evaluated. Here, we demonstrate that pyridostatin exhibits a high specific activity against BRCA1/2‐deficient tumours, including patient‐derived xenograft tumours that have acquired PARP inhibitor (PARPi) resistance. Mechanistically, we demonstrate that pyridostatin disrupts replication leading to DNA double‐stranded breaks (DSBs) that can be repaired in the absence of BRCA1/2 by canonical non‐homologous end joining (C‐NHEJ). Consistent with this, chemical inhibitors of DNA‐PKcs, a core component of C‐NHEJ kinase activity, act synergistically with pyridostatin in eliminating BRCA1/2‐deficient cells and tumours. Furthermore, we demonstrate that pyridostatin triggers cGAS/STING‐dependent innate immune responses when BRCA1 or BRCA2 is abrogated. Paclitaxel, a drug routinely used in cancer chemotherapy, potentiates the in vivo toxicity of pyridostatin. Overall, our results demonstrate that pyridostatin is a compound suitable for further therapeutic development, alone or in combination with paclitaxel and DNA‐PKcs inhibitors, for the benefit of cancer patients carrying BRCA1/2 mutations.

Thank you for the submission of your manuscript to EMBO Molecular Medicine, and please accept my apologies for the delay in getting back to you, as one referee needed more time to complete his/her review. We have now received feedback from the three reviewers who agreed to evaluate your manuscript.
As you will see from the reports below, while referee #3 is overall supportive of publication of the manuscript pending minor revisions, referees #1 and #2 raise serious concerns pertaining both to the limited novelty and to the unsupported mechanism. Referee #1 suggested to turn the manuscript into a short report, that would focus on the activation of the innate immune signaling mechanism and on the novel drug combinations. We would therefore like to invite you to revise the manuscript along these lines, and strengthen the identified mechanism on innate immunity signaling, as well as better define the novelty of the present manuscript in light of previous publications.
Acceptance of the manuscript will entail a second round of review. EMBO Molecular Medicine encourages a single round of revision only and therefore, acceptance or rejection of the manuscript will depend on the completeness of your responses included in the next, final version of the manuscript. For this reason, and to save you from any frustrations in the end, I would strongly advise against returning an incomplete revision.
Revised manuscripts should be submitted within three months of a request for revision; they will otherwise be treated as new submissions, except under exceptional circumstances in which a short extension is obtained from the editor.
When submitting your revised manuscript, please carefully review the instructions that follow below. We perform an initial quality control of all revised manuscripts before re-review; failure to include requested items will delay the evaluation of your revision.
We require: 1) A .docx formatted version of the manuscript text (including legends for main figures, EV figures and tables). Please make sure that the changes are highlighted to be clearly visible.
3) A .docx formatted letter INCLUDING the reviewers' reports and your detailed point-by-point responses to their comments. As part of the EMBO Press transparent editorial process, the point-by-point response is part of the Review Process File (RPF), which will be published alongside your paper. 4) A complete author checklist, which you can download from our author guidelines (https://www.embopress.org/page/journal/17574684/authorguide#submissionofrevisions). Please insert information in the checklist that is also reflected in the manuscript. The completed author checklist will also be part of the RPF.
5) It is mandatory to include a 'Data Availability' section after the Materials and Methods. Before submitting your revision, primary datasets produced in this study need to be deposited in an appropriate public database, and the accession numbers and database listed under 'Data Availability'. Please remember to provide a reviewer password if the datasets are not yet public (see https://www.embopress.org/page/journal/17574684/authorguide#dataavailability).
In case you have no data that requires deposition in a public database, please state so in this section. Note that the Data Availability Section is restricted to new primary data that are part of this study. 6) For data quantification: please specify the name of the statistical test used to generate error bars and P values, the number (n) of independent experiments (specify technical or biological replicates) underlying each data point and the test used to calculate p-values in each figure legend. The figure legends should contain a basic description of n, P and the test applied. Graphs must include a description of the bars and the error bars (s.d., s.e.m.).

7)
We would also encourage you to include the source data for figure panels that show essential data. Numerical data should be provided as individual .xls or .csv files (including a tab describing the data). For blots or microscopy, uncropped images should be submitted (using a zip archive if multiple images need to be supplied for one panel). Additional information on source data and instruction on how to label the files are available at . 8) Our journal encourages inclusion of *data citations in the reference list* to directly cite datasets that were re-used and obtained from public databases. Data citations in the article text are distinct from normal bibliographical citations and should directly link to the database records from which the data can be accessed. In the main text, data citations are formatted as follows: "Data ref: Smith et al, 2001" or "Data ref: NCBI Sequence Read Archive PRJNA342805, 2017". In the Reference list, data citations must be labeled with "[DATASET]". A data reference must provide the database name, accession number/identifiers and a resolvable link to the landing page from which the data can be accessed at the end of the reference. Further instructions are available at . Tables that are collapsible/expandable  -For the figures that you do NOT wish to display as Expanded View figures, they should be bundled together with their legends in a single PDF file called *Appendix*, which should start with a short Table of Content. Appendix figures should be referred to in the main text as: "Appendix Figure S1, Appendix Figure S2" etc.

9) We replaced Supplementary Information with Expanded View (EV) Figures and
-Additional Tables/Datasets should be labeled and referred to as Table EV1, Dataset EV1, etc. Legends have to be provided in a separate tab in case of .xls files. Alternatively, the legend can be supplied as a separate text file (README) and zipped together with the Table/Dataset file.
See detailed instructions here: .
10) The paper explained: EMBO Molecular Medicine articles are accompanied by a summary of the articles to emphasize the major findings in the paper and their medical implications for the non-specialist reader. Please provide a draft summary of your article highlighting -the medical issue you are addressing, -the results obtained and -their clinical impact.
This may be edited to ensure that readers understand the significance and context of the research. Please refer to any of our published articles for an example. 11) For more information: There is space at the end of each article to list relevant web links for further consultation by our readers. Could you identify some relevant ones and provide such information as well? Some examples are patient associations, relevant databases, OMIM/proteins/genes links, author's websites, etc... 12) Every published paper now includes a 'Synopsis' to further enhance discoverability. Synopses are displayed on the journal webpage and are freely accessible to all readers. They include a short stand first (maximum of 300 characters, including space) as well as 2-5 one-sentences bullet points that summarizes the paper. Please write the bullet points to summarize the key NEW findings. They should be designed to be complementary to the abstract -i.e. not repeat the same text. We encourage inclusion of key acronyms and quantitative information (maximum of 30 words / bullet point). Please use the passive voice. Please attach these in a separate file or send them by email, we will incorporate them accordingly. preferable to CX-5461 for future pre-clinical studies.
12. In the Materials and Methods, if the authors developed H1299 cell lines carrying an inducible shBRCA2, more information on this should be given. Alternatively, the commercial source should be given.

Referee #2 (Comments on Novelty/Model System for Author):
Novelty: A previous publication (Xu et al., Nat Commun 2016) reports that another G-quadruplex stabilizer CX-5461 currently in clinical trials is selectively lethal to BRCA1/2 deficient tumors, and also presents an extensive analysis of underlying mechanisms which overlaps with the results & conclusions in this paper. The novelty of the current manuscript is limited.
Medical impact and adequacy of model system: Several factors limit potential impact in molecular medicine. Many of the experiments are carried out using DLD-1 colorectal cancer cells, which is not ideal because these cells are deficient in mismatch repair & accumulate point mutations at a very high rate. The clinical use of PDS is not yet clear, but other G4-stabilizers are already in clinical trials. So, it would be better to repeat at least key experiments using alternative cells & drugs.
Referee #2 (Remarks for Author): In their manuscript entitled "Anti-tumoral activity of the G-quadruplex ligand pyridostatin against BRCA1/2-deficient tumors", Groelly, Porru and colleagues take advantage of various mouse model systems to demonstrate the selective toxicity of the Gquadruplex stabilizer pyridostatin (PDS) towards BRCA1/BRCA2-deficient tumors, including PARP-inhibitor resistant xenografts. In addition, the authors evaluate various drug combinations that act in combination with PDS in targeting BRCA-deficient xenografts in mice. The manuscript is a logical extension of the authors' previous work (Zimmer et al., Mol Cell 2016), confirming and extending their previous in vitro findings.  Fig 5B shows little or no activation of cGAS/STING-induced transcription by BRCA2 shRNA alone. This mechanism needs to be clarified. (c) The authors imply in the abstract and text that paclitaxel potentiates in vivo toxicity of PDS via cGAS-dependent apoptosis. The results shown are not enough to support this mechanism, they only show that the effect of PDS on tumor growth is enhanced by paclitaxel (which has many mechanisms of action). Figure 3 are somewhat surprising. Is the lack of XRCC4 expression in Brca1-/-Tp53bp1-/-cells expected? Is it supported by WB data in whole cell lysates? If that is the case, blots showing whole cell lysates would be more informative.

The results of the cell fractionation experiments in
5. Considerably higher concentrations of PDS and longer drug treatments were used to induce innate immune signalling ( Figure  5) than the conditions used for the other experiments in the manuscript. In fact, levels of PDS that induce substantial DNA damage do not seem to be sufficient to activate the cGAS/STING pathway. This could mean that the mechanisms for cGAS activation by PDS are different from those causing DNA damage.
6. Finally, several factors limit potential impact in molecular medicine. Many of the experiments are carried out using DLD-1 colorectal cancer cells, which is not ideal because these cells are deficient in mismatch repair & accumulate point mutations at a very high rate. The clinical use of PDS is not yet clear, but other G4-stabilizers are already in clinical trials. So, it would be better to repeat at least key experiments using alternative cells & drugs. Minor comments 1. Scattered typos and grammatical mistakes (e.g., "consistent our previously ..." -page 6; "tumors were allowed grow" -page 36; etc.). 2. Some conclusions should be moderated (e.g., on page 7 the authors write: "pyridostatin ... effectively and specifically eliminates ... tumors lacking BRCA2". This is not supported by the data in Fig 1, which shows a lack of tumor growth at best. Xu et al (2016) also show effects of Gq stabilizers on tumors with other DNA repair deficiencies, suggesting the effects are not totally selective. 3. Page 10, last paragraph should refer to Fig 2, not to Fig 1. 4. Page 18, end of first paragraph should read "...47% and 40% increase in survival ..." 5. The antibody list in the Materials and Methods section includes antibodies not used in the manuscript and lacks antibodies shown (e.g., POLQ). 6. Legend for Fig 2G: please delete "BRCA2-proficient (+BRCA2)". 7. Figure 5B: please indicate PDS concentrations. 8. Figure 6D: statistical significance is described in legend but not shown in the figure. 9. Table 4 consists of one line. In general, the Tables might be better suited for supplementary material.
Referee #3 (Remarks for Author): Groelly and Porru et al. provide a comprehensive and systematic study demonstrating the viability of using G4 stabilizers, like pyridostatin (PDS), as an effective chemotherapeutic in an in vivo xenograft model. More importantly, the authors further extend these findings towards selectively targeting BRCA1-mutated xenograft tumors that have developed PARPi resistance, a prevalent and clinically significant outcome when using PARPi to target HR-deficient tumors. Additionally, they demonstrate that pyridostatin in combination with the DNA-PKcs inhibitor, Nu-7441 and microtubule-stabilizing agent, paclitaxel, have potent antitumor activity in BRCA-deficient tumors and propose the use of this combinational therapeutics in treatment of BRCA1/2 mutant tumors. This manuscript provides an important body of work on the potential therapeutic use and effects of PDS, yet some of the conclusions presented in this study are somewhat premature and lack sufficient experimental support, especially with respect to the specific repair pathways utilized in the repair of G4 lesions and would benefit from further investigations. Nevertheless, these do not diminish the overall importance of the data and observations presented in the manuscript, which is of significant interest to the scientific and clinical community at large. I therefore recommend this paper for publication, and ask that the authors address several (minor) points detailed below, and further elaborate in their discussion on possible mechanisms: 1. In previous work by Olivieri et al Cell 2020, the genetic factors involved in DNA damage response were mapped for different therapeutic insults, and categorized according to specific pathways, where PDS was found to be strongly associated with end joining pathway. The Olivieri study and other recent studies (Bruno et al PNAS 2020 -PMCID: PMC7049172; Pipier et al Biorxiv 2020) have linked G-quadruplex ligands to activity of topoisomerase 2, and lesions that are repaired by the NHEJ pathway. On the other hand, drugs that induce replication fork stalling and collapse such as the Topoisomerase I poison CPT2 and PARP inhibitor Olaparib were strongly correlated with HR repair, which is anticipated to facilitate the repair of lesions that occur following fork collapse (rather than end joining). Overall, these studies indicate that lesions induced by PDS will rely on repair via NHEJ whereas lesions induced by PARP inhibition will utilize HR. In the present study, the authors relate PDS treatment to replication fork and use it to target BRCA and HR deficient models, a result that seems to differ from the specific repair pathways that were previously assigned. While I realize that the assignment of repair pathways in Olivieri et al is somewhat lacking with respect to the specific definition of repair mechanisms and the mechanistic complexity of said pathways, I would ask that the authors discuss and at least speculate on the various lesions formed and their repair mechanisms. A possible scenario that the authors might want to consider is that PDS can induce two (or more) types of lesions, one type of lesion in forks that is repaired by HR and another lesion that is repaired by NHEJ. This could be a plausible scenario, considering that PDS might act as a DNA structure driven topoisomerase 2 poison, and the fact that the top2 poison etoposide induces lesions that are either repaired by NHEJ by also HR as demonstrated by Olivieri et al, (in Fig S1B). Such discussion will be very beneficial to readers who might find these results confusing or conflicting, while also providing a conceptual framework for future mechanistic studies. In their discussion, the authors should also consider additional reports that have also used other G-quadruplex ligands to show effect on HR proficient cells (for example PMID: 33173151 and PMID: 32457376) 2. The authors provide an interesting finding that PDS in combination with the DNA-PKcs inhibitor and microtubule-stabilizing agent, paclitaxel, as a therapeutic strategy to target BRCA1-deficient tumors. However, exacerbation of DNA-damage via these methods could potentially have detrimental effects on tumors irrespective of their BRCA1/2 status. I would recommend the assessment of these effects by replicating the experiments in Figure 6C in a BRAC1/2 proficient xenograft tumor model before posing it as a therapeutic strategy specifically for patients with BRCA1/2 mutations. If this is not possible, I would ask that the authors would comments about this matter. 3. Figure 5B: the authors show the error bars, but not if the differences between the samples are significant. What are the associated p-values? 4. Figure 1D lack statistics and smaple size. The authors showed show error bars and reflect the significance and relevant difference between the PDS and Talazoparib. 5. The authors should provide representative images of IF staining for all samples, and particularly of gH2Ax foci (Fsuch as the data in Figure 2F)and 53BP1 foci. These can be included as new SI figures.
We, the authors, are grateful to the Referees for their constructive comments on our manuscript. We have addressed all the points raised by the Referees, which significantly improved our manuscript.

Point-by-point response:
Referee #1: The authors use several xenograft tumor models with specific genetic deficiencies. Although for some experiments it would be useful to examine tumor models with deficiencies for both BRCA1 and BRCA2 (instead of just one), the models are suitable and are utilized in welldesigned experiments.  Tables 1-5. More novel aspects of the current study, which may open new possibilities for the treatment of HR-deficient tumors, including those that are resistant to PARP inhibitors, include the findings that pyridostatin activates innate immune signaling via cGAS-STING, and initial tests of combinations of pyridostatin with paclitaxel and/or NU-7441 to more effectively inhibit the growth of BRCA1-deficient tumors. While many parts of the manuscript seem relatively incremental, these latter aspects are more novel. It is the recommendation of this reviewer, that along with appropriate revisions, this manuscript might be better suited to publication in EMBO Molecular Medicine as a short report that focuses on the activation of innate immune signaling and novel drug combinations. . Therefore, the finding of tumor sensitivity to G4 ligands is not by itself particularly novel. Those studies even use some of the same cell lines, such as DLD1 and HCC116 cells that are deficient for BRCA2. Response: As the Referee points out, other G4 ligands (RHPS4 and CX-5461) have been shown to be active against BRCA1/2-deficient xenograft tumours established in mice. However, these have not yet been successfully used in cancer patients with BRCA mutations, which develop resistance to targeted therapies (e.g. PARP inhibitors; PARPi). Therefore, it is imperative to identify new G4 ligands that not only eliminate BRCA-deficient tumours, but also counteract resistant disease. Here, we demonstrate that pyridostatin inhibits growth of BRCA1/2-deficient xenograft tumours in mice, which recapitulates its activity in vitro. Importantly, pyridostatin has very low in vivo toxicity relative to CX-5461, the only G4-binding compound currently in clinical trials (see answer to point 11 below). This is 23rd Sep 2021 1st Authors' Response to Reviewers a key novel aspect of pyridostatin biology, essential for establishing its translational potential.
2. Xu et al. (2017) show that BRCA1-deficient xenograft tumors developed from cells that are resistant to PARP inhibitor are sensitive to CX-5461. As such, the finding here that BRCA1-deficient/PARP inhibitor resistant tumors are hypersensitive to pyridostatin is not especially novel. Response: The Referee correctly points out that Xu et al. (2017) showed that CX-5461 is active against BRCA1-deficient xenograft tumours established from cells that are resistant to PARP inhibitors (PARPi). Here, we demonstrate that pyridostatin is not only active against tumours established from cells, but also against tumours obtained from patients (PDXs) that have acquired resistance to PARPi. As an additional novel aspect of our work, we demonstrate mechanistically that loss of classical non-homologous end joining (c-NHEJ) repair underlies the sensitivity of BRCA1-deficient PARPi-resistant tumours to pyridostatin.
3. While the authors show that BRCA1/2-deficient tumors treated with pyridostation resume growth when treatment is stopped, and show that this involves c-NHEJ and can be suppressed by the DNA-PKcs inhibitor NU-7441, this is not unexpected since Xu et al. (2017) show that c-NHEJ factors are required for cellular resistance to CX-5461. Response: As the Referee indicates, Xu et al (2017) showed that abrogating c-NHEJ sensitises cells in culture to pyridostatin. However, this does not necessarily imply that abrogating c-NHEJ repair also sensitises BRCA1/2-deficient cells and tumours to pyridostatin, as other backup DNA repair pathways act in the context of BRCA-deficiency and may be involved in the repair of pyridostatin-inflicted damage in these cells. Here we demonstrate for the first time that chemical inhibition of c-NHEJ (using DNA-PK inhibitor NU-7441) inhibits growth of BRCA1/2-deficient cells and tumours and that NU-7441 acts synergistically with pyridostatin and paclitaxel in targeting these tumours. In other words, we investigate whether the DNA damage inflicted by pyridostatin can be repaired in cells lacking homologous recombination (i.e. BRCA1/2-deficient) and, if so, which repair pathways are required. This approach enabled us to define a key resistance mechanism to pyridostatin, uniquely mediated by c-NHEJ, and modalities to counteract it. Moreover, we demonstrate the DNA damage inflicted by pyridostatin in BRCA1/2-deficient cells, including those resistant to PARPi, triggers cGAS/STING dependent innate immune response. These results highlight the potential of pyridostatin treatment to facilitate tumour recognition by the immune system in vivo. Fig. S3B, the authors demonstrate that PRKDC WT and KO HAP1 cells display the same viability over a range of concentrations of NU-7441. This is not convincing evidence that NU-7441 is a specific inhibitor of DNA-PKcs. A better experiment might be to compare the viability of PRKDC KO cells to vehicle vs NU-7441. Response: Following the Referee's suggestion, in the new Figure S4B we represent the viability data in PRKDC KO cells as NU-7441 effect relative to DMSO. Importantly, the specificity of NU-7441 for DNA-PKcs is further demonstrated in Fig. S4D, where NU-7441 potentiates the cytotoxicity of pyridostatin in wild type cells, but not in PRKDC KO cells. 6. The authors utilize male mice for xenograft experiments in Fig. 1/Tables 1-2 and female mice in experiments shown in Fig. 3/ Table 3. At a minimum, there should be a justification for this and perhaps also discussion of how this may affect the results that were obtained. Response: We believe that the exclusive use of male animals would be less informative for future clinical trials and therefore included animals of both sexes in our in vivo experiments. This should strengthen the possible clinical use of pyridostatin for the treatment of BRCA1/2mutated tumours. 7. Innate immune signaling is tested in H1299 cells with DOX-inducible shBRCA2, but no indication is given as to why this system rather than BRCA2-deficient DLD1 and/or HCT116 cells were utilized as elsewhere. Additionally, it might strengthen the manuscript to show activation of innate immune signaling in more than one BRCA2-deficient cell line. Response: We would like to thank the Referee for this suggestion. Consistently, we have included in the new manuscript ( Fig 3D) data showing that pyridostatin induces innate immune responses in BRCA2-deleted RPE-1 cells, which substantially strengthens our findings. 10. The authors should consider whether combinations can be developed in which at least one of the drugs can be continued for maintenance, since this might make the strategy particularly effective. Response: On pages 20-21 of the revised manuscript we discuss whether and how any of the drugs used in the triple combination could be used in maintenance therapy. 11. It might be useful for the authors to consider, in the Discussion, whether there is any reason the use of pyridostatin is preferable to CX-5461 for future pre-clinical studies. Response: We observed that pyridostatin used in mice at the maximum tolerated dose had a higher activity against BRCA1/2-deficient tumours than CX-5461 (at a dose used by Xu et al., 2017). We included these results in the Table for Referees below. In Discussion, we mention that CX-5461, which also acts as an inhibitor of rDNA transcription by preventing RNA polymerase I binding to rDNA promoters (Drygin et al., Cancer Res. 2011), is likely to have adverse effects in patients, as indicated by a recent clinical dose-escalation study (Khot et al., Cancer Disc. 2019). Table for Referees. In vivo anti-tumour efficacy of pyridostatin and CX-5461 on BRCA2 -/-DLD1 and BRCA2 -/-HCT116 xenografts CB17-SCID male mice were were injected intramuscularly with 5x10 6 cells per mouse. Tumours were allowed grow to approximately 250 mm 3 before initiation of treatment (day 1). Mice were treated with with pyridostatin (i.v.; 7.5 mg/kg/day) for five consecutive days, followed by two-day break and five more days of treatment or with CX-5461(o.s.; 50 mg/kg/day) three times, once every three days. Each experimental group included n = 5 mice. Tumour weight inhibition was calculated at the nadir of the effect using the formula: (1 -[tumour weight in treated mice] / [tumour weight in untreated mice]) x100 and expressed as average for n = 5 mice in each group. Tumour growth delay was calculated as the median time in days required for untreated and treated tumours to reach the same size. Stable disease was defined as mice in which tumour volume did not change for at least two weeks after initiation of treatment. Body weight loss is reported as weight at the end of treatment relative to the first day of treatment (%), as average for n = 5 mice in each group. Response: As the Referee correctly points out, some G4 ligands have entered clinical trials, with CX-5461 as a prominent example. Key to their success is good tolerability in patients and effective tumour targeting. Here we propose that pyridostatin is a promising candidate for future clinical development, because it shows low animal toxicity under our experimental conditions (see response to Referee #1, point 11) and efficient targeting of BRCA1/2deficient tumours in mice. In addition to demonstrating the clinical potential for pyridostatin, the work presented in our manuscript is novel and furthers our understanding of G4 ligand biology for the following reasons: 1. Mechanistically, we show that PARPi resistant BRCA1deficient cells lack of c-NHEJ, which is critical for their susceptibility to pyridostatin; 2. We furthermore demonstrate that c-NHEJ provides the main pathway for repair of pyridostatininduced DNA damage in the absence of BRCA1 or BRCA2, and therefore represents a key target for counteracting pyridostatin resistance; 3. Consistent with the former, we demonstrate synergy between pyridostatin and the DNA-PKcs inhibitor NU-7441 against BRCA1/2-deficient tumours; the specific toxicity is further enhanced by the triple combination with paclitaxel; 4. We show that pyridostatin triggers cGAS/STING-dependent immune responses in BRCA1/2-deficient cells, including those that have acquired PARPi resistance, and that this correlates with the pyridostatin ability to inflict ATM-activating DNA damage.  17) we explain that the replication defects and DSBs induced by pyridostatin are inflicted throughout the genome, including the telomeres. We propose that regardless of their position in the genome, DSBs activate the same signalling pathways and are repaired by the same mechanisms.

In
(b) The authors say that PDS activates cGAS/STING in BRCA-deficient cells. Previous reports show that BRCA deficiency itself strongly activates cGAS/STING pathway & downstream signaling. Fig 5B shows little or no activation of cGAS/STING-induced transcription by BRCA2 shRNA alone. This mechanism needs to be clarified. Response: As the Referee points out, our previous work demonstrated activation of the cGAS/STING pathway and downstream innate immune signalling upon BRCA2 abrogation. However, these former experiments were performed under chronic BRCA2 depletion (28 days of DOX treatment; clarified on page 10), in contrast with Fig 3C (formerly Fig 5B) where we used short-term BRCA2 abrogation (3 days of DOX treatment). Under these conditions, and in the absence of genotoxic treatments, innate immune responses are not activated in BRCA2-deficient cells. However, pyridostatin triggers robust cGAS/STING activation in these cells, caused by the rapid increase in DNA damage levels.
(c) The authors imply in the abstract and text that paclitaxel potentiates in vivo toxicity of PDS via cGAS-dependent apoptosis. The results shown are not enough to support this mechanism, they only show that the effect of PDS on tumor growth is enhanced by paclitaxel (which has many mechanisms of action). Response: We agree with the Referee that our data do not show an effect of paclitaxel on cGAS-dependent responses and this is now reflected in the Abstract and text of the revised manuscript. Figure 3 are somewhat surprising. Is the lack of XRCC4 expression in Brca1-/-Tp53bp1-/-cells expected? Is it supported by WB data in whole cell lysates? If that is the case, blots showing whole cell lysates would be more informative. Fig S6A Western blots showing the XRCC4 levels in whole cell extracts prepared from the same cell lines. 5. Considerably higher concentrations of PDS and longer drug treatments were used to induce innate immune signalling ( Figure 5) than the conditions used for the other experiments in the manuscript. In fact, levels of PDS that induce substantial DNA damage do not seem to be sufficient to activate the cGAS/STING pathway. This could mean that the mechanisms for cGAS activation by PDS are different from those causing DNA damage. Response: The Referee questions whether the innate immune signalling triggered by pyridostatin in BRCA2-deficient cells is due to DNA damage accumulation, or to some other, non-specific effect of the treatment. To address this concern, in addition to the results showing dose-dependent induction of DNA damage and immune responses by pyridostatin in BRCA2-deficient cells (Fig 3A)  Minor comments:

Response: In response to the Referee's request, we included in the new
1. Scattered typos and grammatical mistakes (e.g., "consistent our previously ..." -page 6; "tumors were allowed grow" -page 36; etc.). Response: We have corrected these errors in the revised manuscript and are grateful to the Referee for pointing them out.
2. Some conclusions should be moderated (e.g., on page 7 the authors write: "pyridostatin ... effectively and specifically eliminates ... tumors lacking BRCA2". This is not supported by the data in Fig 1, which shows  8. Figure 6D: statistical significance is described in legend but not shown in the figure.

Response:
We have now included a comprehensive statistical analysis of the survival of the different treatment groups in Table S1. 9. Table 4 consists of one line. In general, the Tables might be better suited for supplementary material. Response: To ensure consistency with the other figures/tables showing tumour data, we think it best to keep Table 4 in the main text.

Referee #3
Remarks for Author: Groelly and Porru et al. provide a comprehensive and systematic study demonstrating the viability of using G4 stabilizers, like pyridostatin (PDS), as an effective chemotherapeutic in an in vivo xenograft model. More importantly, the authors further extend these findings towards selectively targeting BRCA1-mutated xenograft tumors that have developed PARPi resistance, a prevalent and clinically significant outcome when using PARPi to target HRdeficient tumors. Additionally, they demonstrate that pyridostatin in combination with the DNA-PKcs inhibitor, Nu-7441 and microtubule-stabilizing agent, paclitaxel, have potent antitumor activity in BRCA-deficient tumors and propose the use of this combinational therapeutics in treatment of BRCA1/2 mutant tumors.
This manuscript provides an important body of work on the potential therapeutic use and effects of PDS, yet some of the conclusions presented in this study are somewhat premature and lack sufficient experimental support, especially with respect to the specific repair pathways utilized in the repair of G4 lesions and would benefit from further investigations. Nevertheless, these do not diminish the overall importance of the data and observations presented in the manuscript, which is of significant interest to the scientific and clinical community at large. I therefore recommend this paper for publication, and ask that the authors address several (minor) points detailed below, and further elaborate in their discussion on possible mechanisms: 1. In previous work by Olivieri et al Cell 2020, the genetic factors involved in DNA damage response were mapped for different therapeutic insults, and categorized according to specific pathways, where PDS was found to be strongly associated with end joining pathway. The Olivieri study and other recent studies (Bruno et al PNAS 2020 -PMCID: PMC7049172; Pipier et al Biorxiv 2020) have linked G-quadruplex ligands to activity of topoisomerase 2, and lesions that are repaired by the NHEJ pathway. On the other hand, drugs that induce replication fork stalling and collapse such as the Topoisomerase I poison CPT2 and PARP inhibitor Olaparib were strongly correlated with HR repair, which is anticipated to facilitate the repair of lesions that occur following fork collapse (rather than end joining). Overall, these studies indicate that lesions induced by PDS will rely on repair via NHEJ whereas lesions induced by PARP inhibition will utilize HR. In the present study, the authors relate PDS treatment to replication fork and use it to target BRCA and HR deficient models, a result that seems to differ from the specific repair pathways that were previously assigned. While I realize that the assignment of repair pathways in Olivieri et al is somewhat lacking with respect to the specific definition of repair mechanisms and the mechanistic complexity of said pathways, I would ask that the authors discuss and at least speculate on the various lesions formed and their repair mechanisms. A possible scenario that the authors might want to consider is that PDS can induce two (or more) types of lesions, one type of lesion in forks that is repaired by HR and another lesion that is repaired by NHEJ. This could be a plausible scenario, considering that PDS might act as a DNA structure driven topoisomerase 2 poison, and the fact that the top2 poison etoposide induces lesions that are either repaired by NHEJ by also HR as demonstrated by Olivieri et al, (in Fig S1B). Such discussion will be very beneficial to readers who might find these results confusing or conflicting, while also providing a conceptual framework for future mechanistic studies. In their discussion, the authors should also consider additional reports that have also used other G-quadruplex ligands to show effect on HR proficient cells (for example PMID: 33173151 and PMID: 32457376) Response: We thank the Referee for suggesting a discussion of the results reported by Olivieri et al. (2020), as well as by other studies, which propose that pyridostatin may also act as a topoisomerase 2 (Top2) inhibitor. Such dual mode of action is not uncommon among G4-binding compounds, as CX-5461 is also known to act as an inhibitor of RNA polymerase I (Drygin et al., Cancer Res. 2011). We are also grateful to the Referee for the excellent suggestion that pyridostatin might induce different types of DNA damage: some derived from G4 stabilisation and others from Topo2 trapping on DNA. Both types of lesions are caused by replication fork stalling at these sites and could be repaired by homologous recombination (HR) or NHEJ. As such, both types of damage should be deleterious to cells lacking BRCA1 or BRCA2. We have included a discussion of this possible scenario on page 18 of our revised manuscript.
2. The authors provide an interesting finding that PDS in combination with the DNA-PKcs inhibitor and microtubule-stabilizing agent, paclitaxel, as a therapeutic strategy to target BRCA1-deficient tumors. However, exacerbation of DNA-damage via these methods could potentially have detrimental effects on tumors irrespective of their BRCA1/2 status. I would recommend the assessment of these effects by replicating the experiments in Figure 6C in a BRAC1/2 proficient xenograft tumor model before posing it as a therapeutic strategy specifically for patients with BRCA1/2 mutations. If this is not possible, I would ask that the authors would comments about this matter. Response: To address this point (also raised by Referee #1, point 9) we have included in the new Fig S7 and Fig S8 in vivo data showing the response of HCT116 BRCA2 +/+ and BRCA2 -/xenografts to the double and triple combinations of pyridostatin, paclitaxel and NU-7441. These new data demonstrate specific toxicity of the pyridostatin/paclitaxel/NU-7441 combinations against HCT116 BRCA2 -/xenograft tumours, leading to increased survival in mice.
3. Figure 5B: the authors show the error bars, but not if the differences between the samples are significant. What are the associated p-values? Response: We have included p-values in the new Fig 3C (previously Fig 5B) showing qRT-PCR data.
4. Figure 1D lack statistics and sample size. The authors should show error bars and reflect the significance and relevant difference between the PDS and Talazoparib. Response: We have repeated the immunohistochemistry (IHC) staining for gH2AX using HCT116 xenograft tumours treated with pyridostatin and included error bars as well as statistical analyses (Fig 1E,F). The analyses done in DLD1 tumours, formerly in Fig 1D, have been moved to Fig S1. 5. The authors should provide representative images of IF staining for all samples, and particularly of gH2Ax foci (such as the data in Figure 2F) and 53BP1 foci. These can be included as new SI figures. Response: As the Referee requested, we included representative images for gH2AX and 53BP1 foci in the new Fig. S2B. Thank you for the submission of your revised manuscript to EMBO Molecular Medicine. I am extremely sorry for this unusual delay, which is due to the fact that we were waiting for the report from ref #2. Unfortunately, despite several chasers, this referee still has not delivered a report, and in order not to delay the process further, we decided to make a decision at this stage. We have received the report from referee #1, who also commented on your responses to referee #3's concerns. Should referee #2 provide a report shortly, we will send it to you, with the understanding that we would not ask you for further-reaching revisions in addition to the ones required in the enclosed report.
As you will see below, while referee #1 acknowledges the effort that was made to respond to the referee's concerns, he/she also regrets that the novelty of this manuscript compared to previous work has not been sufficiently clarified. Therefore, we would like you to revise the manuscript further along the lines suggested by this referee. In particular, we would suggest turning the manuscript into a report, focusing on the original advances presented in this manuscript by comparison with Zimmer et al 2016 and Xu et al 2017, and on the impactful new findings.
As EMBO Press usually encourages one single round of revisions, please be aware that this will be the last chance for you to address these points.
Additionally, please also address the following editorial issues: -Please carefully check the references to your figures in the main text (current Appendix Fig. S6A and S7D are missing), and correct the appendix callouts to "Appendix Table Sx" or "Appendix Fig. Sx") -Please note that the reference list should come before the figure legends, and each reference should list 10 authors max. before et al.
-As part of the EMBO Publications transparent editorial process initiative (see our Editorial at http://embomolmed.embopress.org/content/2/9/329), EMBO Molecular Medicine will publish online a Review Process File (RPF) to accompany accepted manuscripts. This file will be published in conjunction with your paper and will include the anonymous referee reports, your point-by-point response and all pertinent correspondence relating to the manuscript. Let us know whether you agree with the publication of the RPF and as here, if you want to remove or not any figures from it prior to publication. Please note that the Authors checklist will be published at the end of the RPF. Novelty is rated as medium because, although there are clearly novel aspects, there is too much focus, in comparison, on relatively incremental findings. For example, more incremental findings are in main Figures 1-2, while more novel/impactful findings are in supplemental Figures 6-8. The medical impact is also rated as medium because there is not enough focus on the more novel and potentially impactful findings here. The authors often validate their in vitro findings using multiple cell lines with a deficiency for BRCA2 and utilize several xenograft tumor models with specific genetic deficiencies, so the model systems are adequate.
Also, I was asked to comment on whether the authors suitably addressed concerns raised by Reviewer 3 -and I would say yes. Information was added to the Discussion, as requested, to address point 1. The authors added new Fig. S7 and S8 to address whether BRCA2-deficient xenografts respond to combinations that include pyridostatin. Further, the authors added statistics to most figures in response to points 3-4 (but are still missing from Figure S1B), and added representative images of IF staining to new Fig. S2B to address point 5.

Referee #1 (Remarks for Author):
This revised manuscript by Groelly et al. (EMM-2021-14501-V2) clearly made an effort to respond to my comments from the original review of this manuscript. For example, in response to point 5, the authors added a demonstration that NU-7441 is a specific inhibitor of DNA-PKcs using PRKDC KO cells (new Figure S4B), as requested, and also demonstrate that PRKDC is necessary for NU-7441 to potentiate cell killing by pyridostatin (new Figure S4D). Further, in response to point 7, they added a demonstration that pyridostatin induces innate immune responses in a second BRCA2 deficient cell line (new Figure 3D), as suggested. Similarly, the authors added new Figure S6 Fig. 2E? Also, the first paragraph of p. 13 discusses Appendix Fig S4E, but there is no part E in Figure S4. Further, what is the importance of the finding that deficiency for 53BP1 results in an absence of detectable XRCC4 in the chromatin when a defect in C-NHEJ is already suggested by the deficiency for 53BP1?
Dear Lise, thank you very much for the opportunity to revise our manuscript entitled "Anti-tumoral activity of the G-quadruplex ligand pyridostatin against BRCA1/2-deficient tumours" by Groelly, Porru et al. according to the comments of Referee #1.
As outlined in my email and suggested by the Referee, the revised version highlights more clearly in the Introduction and Results sections the clinical benefits of pyridostatin as a second drug with a mechanism of action similar to CX-5461, but superior in terms of toxicity. We also moved Figures 1 and 2 of the previous manuscript to the Appendix and, conversely, moved to the main text previous Appendix Figure S8, detailing the in vivo efficacy of pyridostatin and its combinations in a second tumour model (HCT116 cellderived). Additionally, former Figure 4E showing absence of XRCC4 from chromatin fraction of PARPi-resistant cells (on which the Referee commented), was moved to Appendix Fig  S8 containing immunoblots of the whole cell extracts from the same experiment. The latest text changes are highlighted in blue.
In addition, we addressed all Editorial requests, i.e. placing References before Figure  Legends, corrected reference format and adding the missing references in the text for Appendix Figures S6A and S7D. We agree with RPF online publication.
We hope that with these revisions our manuscript meets the standards for publication in EMBO Molecular Medicine. Many thanks in advance for your consideration and we are looking forward to hearing from you. Thank you for the submission of your revised manuscript to EMBO Molecular Medicine. We have now received the enclosed report from referee #1. As you will see, this referee is still not fully satisfied with the way the data are presented. We have discussed this issue within the team, and as the conclusions are well supported by the data and the technical quality of the manuscript is high (as mentioned by the referees), we think that there is no need to substantially change the manuscript at this point. Therefore, I am pleased to inform you that we will be able to accept your manuscript, once the following minor points will be addressed: 1/ Comments from Referee #1: Please address the issues on manuscript presentation. As mentioned above, we do not ask for a substantial rewriting at this point, but rather for minor modifications that would help the reader understand the medical impact and novelty of this manuscript compared to your previous work. 5/ Thank you for providing The Paper Explained section. I shortened it a bit to fit our format, please let me know if you agree with the following: Problem Mutations in BRCA1 and BRCA2 are frequently found in familial cancers, including breast, ovarian and prostate cancers, as well as in sporadic cancers. Exploiting the vulnerabilities of BRCA1/2-mutated tumours with targeted therapies permits to specifically eliminate these tumours. However, resistance to standard chemotherapeutic regimens and to targeted therapies rapidly develops in patients. Therefore, there is an imperative need to identify novel drug candidates or treatment strategies to treat BRCA1/2-deficient tumours.

Results
We report that the G-quadruplex ligand pyridostatin specifically inflicts DNA damage and eliminates BRCA1/2-deficient tumours in vivo, including PDXs resistant to PARP inhibitors. We demonstrate that, in the absence of BRCA1/2, pyridostatin causes replication fork stalling and DNA double-strand breaks, which can be repaired by C-NHEJ reactions. Furthermore, we show that pyridostatin-inflicted DNA damage leads to formation of cGAS-associated micronuclei, which trigger innate immune responses. Finally, our study demonstrates that pyridostatin is well-tolerated in vivo and that its combination with paclitaxel and the NU-7441 DNA-PKcs inhibitor exhibits long-term anti-cancer activity against BRCA1/2-deficient tumours and substantially increases overall survival in mice. Impact Pyridostatin is a strong candidate drug for targeting BRCA1/2-deficient tumours and for overcoming PARPi resistance in vivo. The combination of this compound with DNA-PKcs inhibitors and paclitaxel could represent an effective treatment for the eradication of BRCA1/2-mutated tumours. Additionally, our results suggest that pyridostatin may potentiate the efficacy of immune checkpoint inhibitors. Thus, pyridostatin has a clear potential for further clinical development.
6/ Thank you for providing a synopsis text and figure. Please make sure that the image has the following dimensions: 550 px wide x 300-600 px high, and that the text remains legible. 7/ As part of the EMBO Publications transparent editorial process initiative (see our Editorial at http://embomolmed.embopress.org/content/2/9/329), EMBO Molecular Medicine will publish online a Review Process File (RPF) to accompany accepted manuscripts. This file will be published in conjunction with your paper and will include the anonymous referee reports, your point-by-point response and all pertinent correspondence relating to the manuscript. Let us know if you don't agree with the publication of the RPF and as here, or if you want to remove any figures from it prior to publication.
I look forward to receiving your revised manuscript. Technical quality is generally high with experiments being conducted in reinforcing cell systems, solid mouse experiments, and appropriate statistical analyses in most instances. While the portions on activation of innate immune signaling by pyridostatin and combination treatments are of interest, issues with the presentation still limit the apparent novelty and medical impact.
Referee #1 (Remarks for Author): In response to previous comments and to better clarify the novelty of the manuscript, the authors have added text at the end of the Introduction and beginning of the Results sections which note that G4 quadruplex stabilizers such as RHPS4 and CX- 5461 have not yet been successfully utilized in patients, so there is a need to test the capacity of other G4 stabilizers, such as pyridostatin. Further, the authors now do a better job at the end of the Introduction of stating the focus here on demonstrating the efficacy of pyridostatin against BRCA-deficient tumors, and also note novel findings that pyridostatin induces innate immune signaling in BRCA1/2-deficient cells and that it is effective against BRCA1-deficient tumors that are resistant to PARP inhibitors. Further, as suggested by this reviewer previously, there was a rearrangement of figures, attempting to make the novelty and importance of the current study more apparent by putting newer and/or impactful results in the main figures, with more confirmatory data as supplemental figures.
Despite these changes, in the opinion of this reviewer, the novelty and importance of this work is still not made sufficiently clear for readers. This is especially so for the non-specialist. Among the contributing factors: 1. It is not until well into the 6th page of the Results section, and only after 6 supplemental figures have been presented, that the first main figure is discussed. Perhaps not surprisingly, much of the Results section feels like there is no central focus. One alternative would be to put a main figure early in the Results section and use it as an anchor on which other main figures build. Supplemental Figures would then be there to support the story. Currently, there appears to be equal weight given to supplemental and main figures, making it more difficult to determine which findings are truly new.
2. The authors appropriately refer to their previous work (Zimmer et al., 2016) multiple times in the Results section as a foundation for discussing results that are shown in this manuscript. However, except for the 3rd paragraph on p. 12 (which begins "The clinical efficacy...") that supports Fig. 2A  Do the data meet the assumptions of the tests (e.g., normal distribution)? Describe any methods used to assess it.

Reporting Checklist For Life Sciences Articles (Rev. June 2017)
This checklist is used to ensure good reporting standards and to improve the reproducibility of published results. These guidelines are consistent with the Principles and Guidelines for Reporting Preclinical Research issued by the NIH in 2014. Please follow the journal's authorship guidelines in preparing your manuscript.

B-Statistics and general methods
the assay(s) and method(s) used to carry out the reported observations and measurements an explicit mention of the biological and chemical entity(ies) that are being measured. an explicit mention of the biological and chemical entity(ies) that are altered/varied/perturbed in a controlled manner. a statement of how many times the experiment shown was independently replicated in the laboratory.
Any descriptions too long for the figure legend should be included in the methods section and/or with the source data.
In the pink boxes below, please ensure that the answers to the following questions are reported in the manuscript itself. Every question should be answered. If the question is not relevant to your research, please write NA (non applicable). We encourage you to include a specific subsection in the methods section for statistics, reagents, animal models and human subjects.

definitions of statistical methods and measures:
a description of the sample collection allowing the reader to understand whether the samples represent technical or biological replicates (including how many animals, litters, cultures, etc.).

The data shown in figures should satisfy the following conditions:
Source Data should be included to report the data underlying graphs. Please follow the guidelines set out in the author ship guidelines on Data Presentation.
Please fill out these boxes ê (Do not worry if you cannot see all your text once you press return) a specification of the experimental system investigated (eg cell line, species name).
Cell line experiments were performed at least three times (unless otherwise stated), with technical triplicates for viability or survival assays. Results show the average of three independent repeats (unless otherwise stated) and SEM bars for the three repeats are shown for every datapoint. Yes. The software Graphpad Prism was used to assess data sets.
Animal studies were randomized.
Investigators were blinded when quantifying microscopy and immunofluoresence images.
All animal analysis was performed blinded to experimental group assignment.

Data
the data were obtained and processed according to the field's best practice and are presented to reflect the results of the experiments in an accurate and unbiased manner. figure panels include only data points, measurements or observations that can be compared to each other in a scientifically meaningful way.

E-Human Subjects
All sources of cell lines and/or references are stated in the Materials and Methods section of the manuscript. All cell lines are routinely tested for mycoplasma contamination and authenticated.